This invention relates to a method and a kit of reagents for the measurement of elastase-1 useful for diagnosis of pancreatic diseases. More particulary, this invention relates to a method for the immunoassy of elastase-1, one of the pancreatic enzymes, contained in body fluid wherein a specific antigen-antibody reaction is used and also to a kit of reagents for such immunoassay.
In recent years, pancreatic diseases show a tendency to increase due to various reasons including diffusion of western-style meals and increase in the consumption of alcoholic drinks and in the number of peoples of advanced age and are now occupying an important position in clinical studies.
The pancreas is located in the deepest position of the peritoneal cavity and it is therefore difficult to find a pancreatic disease or observe the symptoms thereof by way of classical diagnostic methods such as palpation, inspection or auscultation and an X-ray examination. Thus, the development of an effective diagnostic method is strongly demanded in this field.
A blood amylase- or urinal amylase-measuring method is known as a diagnostic method for pancreatic diseases wherein body fluids of patients, chiefly, blood, urine, pancreatic juice, pleural dropsy and abdominal dropsy are used, and has been regarded for the past 20 years as a sole screening method and a definitive diagnostic method for pancreatic diseases. At present, however, this method is not considered to be important for the following reasons:
(1) In the case of chronic pancreatitis, the blood amylase value rarely becomes higher except at the peak stage thereof. On the other hand, the measurement of urinal amylase is of no practical significance and is not suited for diagnosis of chronic pancreatitis. PA1 (2) In the case of acute pancreatitis, both blood amylase and urinal amylase show high values, but, after attack, the period within which such high values are shown is so short that diagnosis of the disease can not be made after expiration of the period. PA1 (3) Since blood amylase is measured as a total quantity of two amylases, i.e. those of salivary and pancreatic origins, the measurement per se of amylase is not deemed as a peculiar testing method for pancreatic diseases. PA1 (1) Because the content of pancreatic elastase-1 i. blood is very small (2 ng/ml), the measurement of the pancreatic elastase-1 by colorimetry is extremely difficult even in the case of using succinyl trialanyl p-nitroanilide (the minimum detection sensibility: 50 ng/ml), the most sensitive synthetic substrate at present. PA1 (2) As elastase inhibitors are present at a concentration of about 10.sup.3 times as much as elastase-1 in blood, the measurement of elastase-1 as enzymatic activity is theoretically impossible. PA1 (3) No assurrance is given for peculiarity in the enzymatic activity-measuring method wherein a substrate is used, since the method is also influenced by enzymes other than elastase-1.
In view of the foregoing reasons, the development of a method for measuring pancreatic enzymes other than amylase is necessary for diagnosis of pancreatic diseases.
Human pancreas or pancreatic juice contains elastase in addition to amylase. The "elastase" is a general term for proteases capable of cleaving elastin, i.e. elastic fibers in connective tissues and is widely distributed in pancreas, luecocytes, thrombocytes, spleen, etc. Two kinds of pancreatic elastase are known which are different from each other in respect of physicochemical and enzymological properties as well as antigenic characteristics. Generally, anionically charged elastase existing in a very small proportion is defined as elastase-1 and cationically charged elastase existing in a very large proportion as elastase-2. These enzymes are quite different in physiological function and have no cross-immunity. Elastase-1 alone is dealt with in the present invention as a pancreatic elastase to be measured for diagnosis of pancreatic diseases. This enzyme is known to have a molecular weight of 30,500, a serum level of 2 ng/ml and a pancreatic juice level of 1.about.30 .mu.g/ml and shows a clear contrast to the other elastase-2 having a molecular weight of 24,500 and a serum level of 71 ng/ml and a pancreatic juice level of 500 .mu.g/ml.
Some attempts have been made hitherto for the purpose of measuring the concentration of blood elastase-1 by way of the enzymatic activity-measuring method. However, the measurement of the quantity of elastase-1 by taking advantage of its enzymatic activity was extremely difficult for the following reasons:
Under the circumstances described above, it was quite impossible to measure the quantity of elastase-1 by way of the conventional enzymatic activity-measuring method. Consequently, there is a great demand for developing a new method for effectively and precisely measuring elastase-1 in place of the conventional enzymatic activity-measuring method, especially in the field of diagnosis of pancreatic diseases.